US20190224124A1 - Methods and Kits for Reducing the Susceptibility of Lipoprotein Particles to Atherogenic Aggregation Induced by Arterial-Wall Enzymes - Google Patents

Methods and Kits for Reducing the Susceptibility of Lipoprotein Particles to Atherogenic Aggregation Induced by Arterial-Wall Enzymes Download PDF

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US20190224124A1
US20190224124A1 US16/328,783 US201716328783A US2019224124A1 US 20190224124 A1 US20190224124 A1 US 20190224124A1 US 201716328783 A US201716328783 A US 201716328783A US 2019224124 A1 US2019224124 A1 US 2019224124A1
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Kevin Jon Williams
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0029—Parenteral nutrition; Parenteral nutrition compositions as drug carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the field of the invention is reducing, in humans, the susceptibility of low-density lipoprotein particles (LDL) and similar particles to aggregation induced by arterial-wall enzymes, such as a sphingomyelinase.
  • LDL low-density lipoprotein particles
  • Similar particles to aggregation induced by arterial-wall enzymes, such as a sphingomyelinase.
  • LDL Low-density lipoprotein particles
  • Atherosclerosis arises from the retention, or trapping, of some fraction of these lipoproteins within the arterial wall, chiefly by their binding to molecules of the extracellular matrix, especially proteoglycans, in the arterial intima (5).
  • the retained atherogenic lipoproteins become modified, and a key modification is aggregation (a process that can also include particle fusion) (6).
  • aggregation a process that can also include particle fusion
  • aggregated apoB-lipoproteins are avidly taken up by local macrophages, loading them with cholesterol, thereby producing “foam cells,” a hallmark of atherosclerosis.
  • aggregation of LDL and related lipoproteins within the arterial wall is a key step in the development and progression of atherosclerosis.
  • sphingomyelinase a deficiency of secretory SMase (a product of the acid sphingomyelinase gene) has been linked to a reduction in LDL retention and atherosclerotic lesions in hypercholesterolemic mice, through effects within the arterial wall, without changing plasma concentrations of apoB-lipoproteins (7).
  • Other arterial-wall enzymes may also contribute, such as other phospholipases (such as a phospholipase A 2 ) and lipoprotein lipase (the latter acting primarily as physical bridge).
  • PLs phospholipids
  • lecithins phosphatidylcholines
  • MLVs multilamellar vesicles
  • unilamellar vesicles meaning vesicles comprised of a single lipid bilayer
  • extrusion e.g., the LIPEX® Extruder
  • high-shear and/or high-pressure methods e.g., Microfluidizer® homogenization technology
  • unilamellar vesicles of at least 50 nm diameter are referred to here as large ‘empty’ vesicles (LEVs), according to common nomenclature, because they do not need to contain an encapsulated drug for the uses herein (nevertheless, encapsulated drugs are also contemplated).
  • LEVs have sometimes also been referred to as large unilamellar vesicles (LUVs), in contrast to small unilamellar vesicles (SUVs), which are typically around 30 nm in diameter.
  • LEVs large unilamellar vesicles
  • SUVs small unilamellar vesicles
  • parenteral administration typically intravenously, to experimental animals or human subjects, even cholesterol-free or cholesterol-poor LEVs at sufficient doses remain as intact particles in the circulation and have the ability to extract cholesterol from peripheral tissues (See discussion in (8)).
  • the primary apolipoprotein of LDL is apoB.
  • Other atherogenic lipoprotein particles that contain apoB are also susceptible to SMase.
  • the beneficial effect of LEVs on the susceptibility of LDL to aggregate when exposed to SMase can be expected to occur also with those other atherogenic particles that contain apoB (collectively, LDL and these other atherogenic particles are sometimes referred to as ‘apoB-containing lipoproteins’ or more simply ‘apoB-lipoproteins’).
  • C-TRLs very low-density lipoprotein
  • VLDL very low-density lipoprotein
  • sVLDL small VLDL
  • cholesterol-rich remnant lipoproteins ß-VLDL, VLDL remnants, chylomicron remnants, postprandial remnants
  • IDL intermediate-density lipoprotein
  • lipoprotein(a) [Lp(a)] lipoprotein(a) [Lp(a)]
  • TRLs triglyceride-rich remnant lipoproteins
  • chylomicrons Although chylomicrons also contain apoB, chylomicrons are generally too large to start with to efficiently enter the arterial wall and cause atherosclerosis (see Borén J and Williams K J. The central role of arterial retention of cholesterol-rich apoB-containing lipoproteins in the pathogenesis of atherosclerosis: a triumph of simplicity Curr Opin Lipidol. 2016, in press. doi: 10.1097/MOL.0000000000000330).
  • LEVs have advantages compared to MLVs and SUVs.
  • MLVs and SUVs By having only a single phospholipid bilayer, most of the lipid content of LEVs is directly exposed, i.e., available to beneficially alter LDL and other apoB-lipoproteins.
  • the multilamellar structure of MLVs means that internal bilayers are shielded and therefore less efficient at altering LDL and other atherogenic lipoproteins to become less susceptible to aggregation.
  • SUVs have a harmful side-effect of suppressing LDL receptor expression in the liver, thereby increasing plasma concentrations of LDL (8).
  • LEVs avoid the side-effect of suppressing LDL receptors and hence the side-effect of raising plasma concentrations of LDL (8).
  • the present inventions are also relevant to the formation of crystals of unesterified cholesterol (“cholesterol crystals”) and other harmful materials within the arterial wall.
  • Such other harmful materials include, but are not limited to dangerous lipids and lipid-rich structures, modified apoB 100 and apoB 48 and their fragments.
  • Additional maladaptive responses to cholesterol from retained and aggregated apoB-lipoproteins include abnormal unesterified cholesterol-enrichment of cell membranes 26 that then activates phagocytic pathways, 28 toll-like receptors, 29 the inflammasome 30 and enzymes that produce pro-retentive arterial matrix. 31-33
  • apoB activates proatherogenic T-cell hybridomas (as indicated by release of IL2, [ 3 H]thymidine incorporation, and other known methods).
  • LEVs can be employed to inhibit the aggregation of apoB-lipoproteins and therefore counter the formation of cholesterol crystals, abnormal cholesterol-enrichment of cell membranes, denaturation of apoB, and the development of other harmful materials derived from apoB-lipoproteins aggregated in the presence of sphingomyelinase.
  • the present invention addresses the need for methods and compositions to target the initial steps in provoking these maladaptive immune responses.
  • the present invention avoids side-effects, including immune suppression and other immune derangements, that arise from current methods to inhibit IL1ß, IL6, and other immune mediators or functions.
  • 24, 25, 36, 37 For example, a recent clinical trial showed that an inhibitor of IL1ß administered to cardiovascular patients was associated with a higher incidence of fatal infection in these patients than was placebo.
  • 25, 36, 37 Moreover, current methods directed towards suppressing immune functions fail to address the root cause of apoB-lipoprotein aggregation and retention, and the formation of cholesterol crystals, abnormally cholesterol-enriched membranes, denatured apoB, and other harmful lipoprotein-derived material.
  • the present invention is disease-specific, i.e., directed to processes that occur in the initiation, progression, and destabilization of atherosclerotic plaques.
  • the present invention represents a major advance in addressing the clinical problem of residual or unrecognized cardiovascular risk.
  • FIG. 1 is a graph showing the relationship of mean aggregate size in nanometers of LDL particles (vertical axis) to the number of hours (horizontal axis) that purified LDL preparations were incubated with SMase and allowed to aggregate. (Nearly all of the enzymatic digestion of sphingomyelin is expected to occur early on; the result is a change in the conformation of apoB that leads to gradual aggregation during the next 18-24 hours—see references (9) and (10).)
  • FIG. 2 shows the data from 0 to 5 hours from FIG. 1 , using expanded horizontal- and vertical-axis scales.
  • FIG. 3 shows the mean diameter and narrow size distribution, as assessed by dynamic light scattering, of a typical preparation of POPC LEVs made by extrusion.
  • the horizontal axis shows the diameter of the particles in nanometers (nm).
  • the count rate was 235.2 kilo counts per second (kcps).
  • the “Z-average diameter size” was 110.8 nm.
  • the polydispersity index (“PDI”) was 0.041, and the PDI width was 22.38 nm.
  • the mean diameter of the peak area was 116.9 nm.
  • FIG. 4 shows the decrease in the molar ratio of sphingomyelin to phosphatidylcholine (SM:PC) in the LDL of LEV-treated mice compared with LDL of saline-treated mice.
  • the invention is the administration of LEVs to humans (and other animals) to decrease the susceptibility of LDL and related lipoproteins to form aggregates. Aggregates of these lipoproteins are key contributors to intra-arterial accumulation of cholesterol and other harmful material and hence the formation of atherosclerotic plaques that cause heart attacks, strokes, peripheral vascular disease, and other forms of atherosclerotic cardiovascular disease (ASCVD).
  • ASCVD atherosclerotic cardiovascular disease
  • the LEVs are administered in order to inhibit the formation of crystals of unesterified cholesterol, abnormal cholesterol-enrichment of cell membranes, denaturation of apoB, the development of other harmful materials derived from apoB-lipoproteins aggregated in the presence of sphingomyelinase, inflammasome activation (particularly the NLRP3 inflammasome), activation of proatherogenic T-cells, release of harmful cytokines (such as IL13 and IL6), plaque progression and destabilization, and release of C-reactive protein (“CRP”).
  • CRP C-reactive protein
  • a human is considered herein to be an “animal.”
  • ApoB refers to apolipoprotein-B, a term that comprises both the full-length form, apoB 100 , and the truncated form, apoB 48 .
  • LDL low-density lipoprotein particles
  • S-SMase refers to sphingomylenase. As used herein it is a general abbreviation for all sphingomyelinases. The main sphingomyelinase in the arterial wall involved in atherosclerotic plaque development is the secretory SMase (“S-SMase”).
  • VLDL refers to very-low-density lipoprotein particles.
  • IDL refers to intermediate-density lipoprotein particles.
  • Lp(a) refers to lipoprotein(a), a form of LDL that includes the apolipoprotein(a).
  • C-TRL refers collectively to cholesterol- and triglyceride-rich apoB-containing lipoproteins, a group that comprises, in particular, cholesterol- and triglyceride-rich apoB-containing remnant lipoproteins.
  • TRL refers to triglyceride-rich lipoproteins, a group that comprises triglyceride-rich apoB-containing remnant lipoproteins.
  • ⁇ -VLDL refers specifically to a type of cholesterol-rich remnant lipoprotein particle seen in type III dyslipoproteinemia and in apoE knock-out mice.
  • VLDL small VLDL
  • sVLDL particles refers to small very-low-density lipoprotein particles.
  • LEV stands for “large empty vesicle.” LEVs have also been referred to as “LUV,” which stands for large unilamellar vesicle. The terms “LUV” and “LEV” are used interchangeably.
  • POPC palmitoyloleoylphosphatidylcholine a/k/a 1-palmitoyl, 2-oleyl phosphatidylcholine, a/k/a palmitoyl-oleoyl phosphatidyl choline.
  • vesicle and “liposome” are used interchangeably in this document.
  • Atherogenic lipoprotein particle refers to atherogenic apolipoprotein particles that comprise apolipoprotein B.
  • TG-rich apoB-lipoproteins refers to atherogenic TG-rich apoB-lipoproteins.
  • the invention is a method (“the method of the invention”) of decreasing the susceptibility of atherogenic lipoprotein particles to aggregation induced by a sphingomyelinase (SMase) in an animal that comprises a SMase and said atherogenic lipoprotein particles, said method comprising administering vesicles (or liposomes) to said animal so as to cause a decrease in said susceptibility, provided said vesicles or liposomes do not comprise significant amounts of sphingomyelin or unesterified cholesterol, and wherein a human is considered to be an animal, and wherein the animal comprises a closed circulatory system that comprises an artery.
  • SMase sphingomyelinase
  • SM:PL sphingomyelin:phospholipid
  • Do not comprise significant amounts as related to unesterified cholesterol will, on the average, means that the vesicles or liposomes comprise unesterified cholesterol to a level such that that the unesterified cholesterol: phospholipid (UC:PL) molar ratio in the LEVs is below 0.1.
  • UC:PL molar ratio is not more than 0.05, more preferably not more than 0.01, even more preferably not more than 0.003, and most preferably not more than 0.001.
  • the method is not applied to a human with dyslipidemia.
  • a human with dyslipidemia Of interest would be a person with ASVCD who is receiving therapy with a statin, ezetimibe, and/or a PCSK9 inhibitor and has achieved therapeutic targets for LDL or apoB concentrations in plasma. That person may no longer have a dyslipidemia, yet still has atherosclerotic plaques and likely still has residual cardiovascular risk. Therefore, of particular interest is an individual at high risk (recognized or unrecognized) of an ASCVD event but who at the moment no longer has a dyslipidemia, owing to successful LDL-lowering therapies.
  • the method of the invention is applied to a human.
  • the method of the invention is applied to a human at (moderate, high, or very high) atherosclerotic cardiovascular risk.
  • a human can be identified by the presence of one or more characteristics selected from the group consisting of known presence of atherosclerotic cardiovascular disease (ASCVD; for example as indicated by a ASCVD risk calculator), high plasma concentrations of LDL, high plasma concentrations of apoB, high plasma concentrations of an apoB-lipoprotein, high blood pressure, history of high blood pressure, smoking, history of smoking, diabetes mellitus, the metabolic syndrome, components of the metabolic syndrome, the atherometabolic syndrome, a high plasma concentration of C-reactive protein, a high coronary artery calcium score, an abnormal carotid ultrasound, an imaging method indicating vulnerable plaque, an imaging method showing macrophage activation in the arterial wall, an imaging method showing protease activity in the arterial wall, and an assay showing high susceptibility of LDL or other apoB-lipoproteins to aggregation and/
  • the method is not applied to a human with dyslipidemia.
  • the aforementioned “orphan or common disease” that predisposes a human to ACSVD can be selected from the group consisting of familial hypercholesterolemia, heterozygous familial hypercholesterolemia, homozygous familial hypercholesterolemia, ‘polygenic’ familial hypercholesterolemia, type IIa hyperlipidemia, type IIb hyperlipidemia, type III hyperlipidemia, type IV hyperlipidemia, a disease caused by a recessive, co-dominant, or dominant mutation that causes hypercholesterolemia, combined hyperlipidemia, familial combined hyperlipidemia (FCHL), a condition with high plasma concentrations of Lp(a), and a condition with high plasma concentrations of apoB. Also contemplated is a condition associated with increased susceptibility of plasma LDL and/or other apoB-lipoproteins to aggregation upon exposure to SMase.
  • a subset of those orphan or common diseases are familial hypercholesterolemia, heterozygous familial hypercholesterolemia, homozygous familial hypercholesterolemia, ‘polygenic’ familial hypercholesterolemia, type IIa hyperlipidemia, type IIb hyperlipidemia, type III hyperlipidemia, type IV hyperlipidemia, a disease caused by a recessive, co-dominant, or dominant mutation that causes hypercholesterolemia, combined hyperlipidemia, and familial combined hyperlipidemia (FCHL).
  • familial hypercholesterolemia heterozygous familial hypercholesterolemia, homozygous familial hypercholesterolemia, ‘polygenic’ familial hypercholesterolemia, type IIa hyperlipidemia, type IIb hyperlipidemia, type III hyperlipidemia, type IV hyperlipidemia, a disease caused by a recessive, co-dominant, or dominant mutation that causes hypercholesterolemia, combined hyperlipidemia, and familial combined hyperlipidemia (FCHL).
  • apoB apoB
  • Plasma concentrations of apoB considered to be higher than desirable or recommended depend on cardiovascular risk; it is generally known in the art that apoB levels at ⁇ 80 and ⁇ 100 mg/dL can be reasonable goals for subjects with very high and high CV risk, respectively (11 at 2352).
  • an apoB level of 100 mg/dL or higher is considered higher than normal.
  • ACSVD disease a condition associated with higher than normal susceptibility of plasma LDL and/or other apoB-lipoproteins to aggregation upon exposure to SMase.
  • the vesicles are administered parenterally.
  • the vesicle is an LEV.
  • the vesicles comprise one or more phospholipids, provided the vesicles do not comprise significant amounts of sphingomyelin.
  • the vesicles comprise a phospholipid that is selected from the group consisting of phosphatidylcholine (especially egg phosphatidylcholine), phosphatidylglycerol (especially egg phosphatidylglycerol), distearoylphosphatidylcholine, distearoylphosphatidylglycerol, and POPC.
  • the atherogenic lipoprotein particle whose susceptibility to aggregation induced by SMase comprises apolipoprotein B.
  • Those particles are preferably selected from the group consisting of LDL, remnant lipoproteins, cholesterol- and triglyceride-rich remnant lipoproteins (together, referred to C-TRLs), very low-density lipoprotein (VLDL), small VLDL (sVLDL), cholesterol-rich remnant lipoproteins, ß-VLDL, VLDL remnants, chylomicron remnants, postprandial remnants, intermediate-density lipoprotein (IDL), lipoprotein(a) [Lp(a)], and triglyceride-rich lipoproteins (TRL).
  • apolipoprotein B refers to the full-length apopB 100 (secreted mostly from the liver in humans), as well as the truncated apoB 48 (secreted mostly from the intestine in humans).
  • LDL are particles of particular interest. It is understood that these lipoproteins can aggregate with their own kind and/or with other apoB-lipoproteins, e.g., LDL can aggregate with LDL, LDL particles can also make a mixed aggregate with C-TRLs, and so forth. Likewise, C-TRLs can aggregate with each other.
  • the total vesicle dose administered per kg of body weight of the human is in the range 10 mg/kg to 1600 mg/kg (preferably in the range 100 to 1600 mg/kg, most preferably in the range 300 mg/kg to 1000 mg/kg), said total dose either administered as a single dose or divided into multiple doses, wherein said multiple divided dosages are administered over at most a short time period (such as 24 hours); and wherein said total vesicle dose is administered at least once.
  • the susceptibility to aggregation of the atherogenic lipoprotein particles and/or their retention by arteries is determined using an assessment system, said assessment capable of measuring such susceptibility and/or retention.
  • assessment systems are described in detail below in the section “Assessment system for measuring the susceptibility to aggregation of the atherogenic lipoprotein particles and/or their retention by arteries”.
  • the invention is a method of measuring susceptibility of atherogenic lipoprotein particles to aggregation induced by a sphingomyelinase (SMase) in a human or other animal, said method comprising the steps of (1) obtaining a sample of plasma from a human or other animal to whom vesicles or liposomes have been administered; and (2) subjecting that sample to a test for susceptibility of its atherogenic lipoprotein particles to aggregation induced by a SMase; wherein said vesicles or liposomes do not comprise significant amounts of sphingomyelin or unesterified cholesterol.
  • SMase sphingomyelinase
  • the time between step (1) and the start of step (2) is preferably not more than 7 days, more preferably not more than 3 days, most preferably not more than one day.
  • the plasma sample is preferably stored at not more than ambient temperature (e.g., about 25 degrees centrigrade (° C.) in the interval between step (1) and the start of step (2).
  • the method is extended to comprise a step of modifying the vesicle (e.g., LEV) dose based on the results obtained using the assessment in the human or other animal such that if a dose (a reference dose) leads to a result selected from the group consisting of less aggregation, a change in atherogenic lipoprotein particle composition indicating less aggregation susceptibility, less retention in an arterial wall, an assessment of an adverse response in an artery to aggregated LDL or other apoB-lipoproteins, such as macrophage accumulation, activation, or M1 polarization, and/or expression of a protease, protease activity, tissue factor, or atherogenic cytokine, then the next LEV dose is decreased compared to the reference dose and/or the time interval between the reference dose and the next dose is increased compared to the time interval between the reference dose and the previous dose.
  • a dose e.g., LEV
  • the next LEV dose is decreased compared to the reference dose and/or the time
  • a dose is as discussed above—either a single dose or multiple doses administered over at most a short time period. Failure of an LEV dose to result in sufficiently less aggregation, less aggregation susceptibility, less retention, and/or an assessment of an adverse response in an artery to aggregated LDL or other apoB-lipoproteins, such as macrophage accumulation, activation, or M1 polarization, and/or expression of a protease, protease activity, tissue factor, or atherogenic cytokine indicates that an increase in dosage (higher amount and/or more frequent administration) should be considered.
  • an aspect of the invention is a method of modifying a vesicle dose in a human or other animal, said method comprising the steps of:
  • a dose (“reference dose”) of vesicles or liposomes to a human or other animal so as to change the susceptibility, in said human or other animal, of atherogenic particles to SMase-induced aggregation;
  • assessing a result in said human or other animal based on a result obtained using an assessment system, said result selected from the group consisting of less aggregation, a change in atherogenic lipoprotein particle composition indicating less aggregation susceptibility, less retention in an arterial wall, an assessment of an adverse response in an artery to aggregated LDL or other apoB-lipoproteins, such as macrophage accumulation, activation, or M1 polarization, and/or expression of a protease, protease activity, tissue factor, or atherogenic cytokine; and
  • the method is not applied to a human with dyslipidemia.
  • the liposomes or vesicles are administered with another medication. Such possible medications are discussed below.
  • the method of the invention is used to effect at least one change in the composition of the LDL (or other apoB-lipoprotein) of the human or other animal, said change selected from the group consisting of a decrease in the molar ratio of sphingomyelin to phosphatidylcholine (SM:PC), an increase in the molar fraction of PC that is POPC, a decrease in the ratio of unesterified cholesterol to phosphatidylcholine (UC:PC), a decrease in the lysoPC:PC ratio, an increase in the ratio of PC:protein, an increase in the ratio of POPC:protein, an increase in the ratio of PC to apoB, an increase in the ratio of POPC to apoB, an increase in the ratio of PC to cholesteryl ester (PC:ChE), an increase in the ratio of POPC:ChE, an increase in the ratio of PC to triglycerides (PC:TG), an increase in the ratio of POPC:TG, a decrease in the UC:protein ratio
  • the vesicle or liposome used in the method comprises a phospholipid that is the same as the phospholipid whose molar fraction will be increased in the LDL or other apoB-lipoprotein.
  • the invention is a kit (“the kit of the invention”).
  • the kit is for decreasing the susceptibility of atherogenic lipoprotein particles to aggregation in a human (or other animal), said kit comprising:
  • the kit is intended for decreasing the susceptibility of atherogenic lipoprotein particles to aggregation induced by SMase and that may be specified in the printed notice.
  • the vesicle is an LEV.
  • the vesicles comprise one or more phospholipids, provided the vesicles do not comprise significant amounts of sphingomyelin or unesterified cholesterol.
  • the vesicles comprise a phospholipid that is selected from the group consisting of phosphatidylcholine (especially egg phosphatidylcholine), phosphatidylglycerol (especially egg phosphatidylglycerol), distearoylphosphatidylcholine, distearoylphosphatidylglycerol, POPC, combinations thereof, and derivatives thereof.
  • POPC is a highly preferred phospholipid.
  • the kit is intended to reduce the SMase-induced aggregation of atherogenic lipoprotein particles: LDL, remnant lipoproteins, cholesterol- and triglyceride-rich remnant lipoproteins (together, referred to C-TRLs), very low-density lipoprotein (VLDL), small VLDL (sVLDL), cholesterol-rich remnant lipoproteins, ß-VLDL, VLDL remnants, chylomicron remnants, postprandial remnants, intermediate-density lipoprotein (IDL), lipoprotein(a) [Lp(a)], and triglyceride-rich remnant lipoproteins (TRL).
  • VLDL very low-density lipoprotein
  • sVLDL small VLDL
  • IDL intermediate-density lipoprotein
  • IDL intermediate-density lipoprotein
  • lipoprotein(a) [Lp(a)] lipoprotein(a)
  • TRL triglyceride-rich remnant lipoproteins
  • apo- refers to a protein component of a lipoprotein, e.g., apolipoproteins can be isolated after the lipid of the lipoprotein has been removed.
  • the printed notice may be on sheet of paper, a label, or a package.
  • the printed notice requirement of the kit of the invention is satisfied if the kit comprises a printed notice of where the user can go (for example to a website) to find out that the kit can be used to decrease the susceptibility of atherogenic lipoprotein particles to aggregation in a human (or other animal with a closed circulatory system).
  • the kit of the invention is combined with an assessment system for measuring extent of aggregation of atherogenic lipoprotein particles and/or their retention in an artery, arteries, or arterial segment of a human or other animal.
  • assessments are discussed above in relation to the methods of the invention, specifically as to systems that can be used to determine whether the LEV dose should be modified.
  • kit comprises an assessment system
  • kit can be referred to as a system of the invention.
  • the inventions are methods in which the vesicles (or liposomes) are administered in order to inhibit the formation of crystals of unesterified cholesterol, abnormal cholesterol-enrichment of cell membranes, denaturation of apoB, the development of other harmful materials derived from apoB-lipoproteins aggregated in the presence of sphingomyelinase, inflammasome activation (particularly the NLRP3 inflammasome), activation of proatherogenic T-cells, release of harmful cytokines (such as IL1 ⁇ and IL6), plaque progression and destabilization, and release of C-reactive protein (“CRP”).
  • the inventions are methods of monitoring the efficacy of those methods. These effects are achieved without harmful immune suppression or other harmful immune derangements.
  • the aforementioned methods of monitoring efficacy include, but are not limited to, assays of apoB-lipoprotein accumulation within the arterial wall, apoB-lipoprotein aggregation within the arterial wall, cholesterol crystal formation within the arterial wall, inflammasome activation, inflammasome activation within the arterial wall, T cell activation, T cell activation within the arterial wall, release of harmful cytokines such as active IL1ß and IL6, release of IL2, and levels of the marker CRP.
  • An example of an assay for apoB-lipoprotein accumulation within the arterial wall is administration of labeled lipoproteins then assessment of the accumulation of their label within the arterial wall.
  • An example of an assay for apoB-lipoprotein aggregation within the arterial wall is administration of doubly labeled lipoproteins such that their aggregation either quenches or enhances the label.
  • An example of an assay for cholesterol crystal formation within the arterial wall is administration of labeled lipoproteins such that cholesterol nucleation enhances the signal (as in Guarino et al. 2004).
  • an assay for inflammasome activation is the release of related cytokines and downstream products, such as IL1ß, IL6, and CRP.
  • An example of an assay for inflammasome activation within the arterial wall is inflammasome-specific imaging.
  • An example of an assay for T-cell activation is the release of T-cell-specific cytokines.
  • An example of an assay for T-cell activation within the arterial wall is T-cell-specific imaging.
  • An example of an assay for release of harmful cytokines is a quantitative assay of their concentrations in plasma or serum or other body fluids.
  • the susceptibility to aggregation of the atherogenic lipoprotein particles and/or their retention by arteries is assessed in an assessment system.
  • the susceptibility to aggregation of the atherogenic lipoprotein particles and/or their retention by arteries is assessed in an assessment system that forms part of the system of the invention.
  • such an assessment system is an assay in vitro and/or a system for assessment of those particles in vivo, and/or by an assay of the level of aggregation indicators in the human or other animal of interest.
  • the assay in vitro (or ex vivo) for assessment of susceptibility to aggregation is preferably selected from the group consisting of (1) measurement of the extent or rate of aggregation of LDL that has been isolated from plasma and then incubated ex vivo with an SMase (a/k/a the susceptibility of said LDL to aggregation induced by SMase), (2) the aggregation of apoB-lipoproteins isolated from plasma and then incubated ex vivo with an SMase, (3) the aggregation of LDL or another apoB-lipoprotein isolated from plasma and then incubated with an arterial-wall enzyme, (4) the aggregation of apoB-containing lipoproteins still in plasma isolated from the human or other animal or with other plasma components, (5) the aggregation of apoB-containing lipoproteins in the presence of plasma components to which one adds an enzyme such as an arterial wall enzyme, (6) the aggregation of apoB-lipoproteins by
  • a system for the determination of the composition of an apoB-lipoprotein would be any system that can determine the relative concentrations of the components of an apoB-lipoprotein. Susceptibility can be inferred from such a determination.
  • the arterial-wall enzyme used in the assay in vitro for assessment of susceptibility to aggregation is preferably selected from the group consisting of a SMase, a human SMase, a human recombinant SMase, a SMase used at an acidic pH, a phospholipase, a phospholipase A 2 , a lipase, a cholesteryl esterase, a lysosomal acid lipase, a protease, a matrix metalloproteinase, a caspase, a furin, an intracellular protease, a calpain, the proteasome, a cathepsin, an extracellular protease, an intracellular hydrolase that is released from a cell.
  • assays of LDL aggregability or composition can be automated, such as on a clinical autoanalyzer, automated mass spectrometry, nephelometry, ELISA and ELISA-like assays, turbidometric analyses, rate-zonal centrifugation, and/or dynamic light scattering (DLS).
  • LDL aggregability or composition or of other atherogenic lipoproteins
  • DLS dynamic light scattering
  • the system for assessment of the retention of the atherogenic particles in the arteries in vivo is preferably selected from the group consisting of an assay of apoB-lipoprotein aggregation and/or retention within the arterial wall in vivo, an imaging method of retained and/or aggregated lipoproteins within the arterial wall, an assay of lipoprotein aggregation and/or retention in a healthy arterial segment, an assay of lipoprotein aggregation and/or retention in a diseased arterial segment, an imaging method (such as cardiac catheterization, intravascular ultrasound (IVUS), an MRI, an MRI with contrast, a CT scan, a scan with contrast, an imaging method with a contrast agent wherein said contrast agent comprises a nanoparticle, and a nuclear medicine study), a method that involves injection of said apoB-lipoprotein into an animal, a method that involves labeling of said apoB-lipoprotein followed by its injection into an animal (said animal comprising a human and a non-human animal), and a method that involves assessments
  • any amphipathic material that allows a liposomal or micellar structure can be used to make the LEVs.
  • Phospholipids are a preferred material. Inclusion of significant amounts (defined above) of sphingomyelin or unesterified cholesterol in the LEVs, however, is to be avoided, and that fact should be understood as a caveat in all discussions of LEV structure and composition herein.
  • Preferred phospholipids for use in formation of LEVs are phosphatidylcholine (especially egg phosphatidylcholine), phosphatidylglycerol (especially egg phosphatidylglycerol), distearoylphosphatidylcholine, distearoylphosphatidylglycerol, palmitoyl-oleoyl phosphatidyl choline (POPC), dimyristoylphosphatidylcholine, soybean phosphatidylcholine, soybean phosphatidylglycerol, lecithin, P,y-dipalmitoyl-a-lecithin, phosphatidylserine, phosphatidic acid, N(2,3di(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimethylammonium chloride, phosphatidylethanolamine, lysolecithin, lysophosphatid
  • highly preferred phospholipids for formation of LEVs are phosphatidylcholine (especially egg phosphatidylcholine), phosphatidylglycerol (especially egg phosphatidylglycerol), distearoylphosphatidylcholine, distearoylphosphatidylglycerol, POPC, combinations thereof, and derivatives thereof.
  • a very highly preferred phospholipid is POPC and, accordingly, LEVs that comprise POPC are very highly preferred.
  • phospholipid molecules that comprise phosphatidyl choline as part of their chemical composition are a preferred group.
  • the POPC or other phospholipid LEV component can be supplemented with small amounts of other lipids or molecules, such as sphingosine-1-phosphate (S1P) and/or specific classes of lysoPC that interfere with SMase-induced aggregation of LDL and related lipoprotein.
  • S1P sphingosine-1-phosphate
  • lysoPC specific classes of lysoPC that interfere with SMase-induced aggregation of LDL and related lipoprotein.
  • the liposomes may also be bound to a variety of proteins and polypeptides to increase the remodeling of endogenous LDL. Binding of apolipoproteins (apoproteins) to the liposomes is particularly useful. As used herein, “bound to liposomes” or “binding to liposomes” indicates that the subject compound is covalently or non-covalently bound to the surface of the liposome or contained, wholly or partially, in the interior of the liposome. Apoprotein A-I (apoA-I), apoprotein A-II (apoA-II), and apoprotein E (apoE) will generally be the most useful apoproteins to bind to the liposomes.
  • apoA-I apoprotein A-II
  • apoE apoprotein E
  • amphipathic, exchangeable apoproteins inhibit the aggregation of LDL and related atherogenic apoB-containing lipoproteins.
  • ApoA-I mimetic peptides such as the 4F peptide, are similarly of use.
  • Other amphipathic peptides are of similar use in this invention.
  • Liposomes used in the methods, kits or systems of the present invention may be bound to molecules of apoprotein A-I, apoprotein A-II, lecithin-cholesterol acyltransferase, and/or small amphipathic peptides (such as apolipoprotein A-I mimetic peptides, the 4F peptide, and/or peptides that mimic amphipathic sequences from proteins or apoproteins, such as apoA-I, apoE, an apoC, apoJ, apoM, and apoB), singly or in any combination and molar ratio.
  • small amphipathic peptides such as apolipoprotein A-I mimetic peptides, the 4F peptide, and/or peptides that mimic amphipathic sequences from proteins or apoproteins, such as apoA-I, apoE, an apoC, apoJ, apoM, and apoB
  • Additional proteins or other non-protein molecules may also be useful to bind to the liposomes to enhance liposome stability, half-life, and other properties, as well as remodeling of LDL and related apoB-lipoproteins.
  • additional proteins or other non-protein molecules include, without limitation, polyethyleneglycol, alkylsulfates, ammonium bromide, and albumin. (The term, “without limitation”, means that there are other such proteins or other non-protein molecules beyond those listed.)
  • Non-phosphorus containing lipids may also be used in the liposomes of the compositions of the present invention. These include, without limitation, stearylamine, docecylamine, acetyl palmitate, and fatty acid amides. Additional lipids suitable for use in the LEVs of the present invention are well known to persons of skill in the art and are cited in a variety of well-known sources (see, for example, reference 12).
  • the LEV preparation can be supplemented for purposes of sterility and stability with compounds used with other drug preparations that are to be administered intravenously (iv).
  • synthetic, non-allergenic phospholipids are preferable to naturally occurring phospholipids.
  • synthetic POPC is preferable over egg PC.
  • LEVs made from phosphatidyl choline have been successfully used in previous experiments related to cholesterol transport from peripheral tissue to the liver. (See discussion in reference (8)). Their production has been described in Rodrigueza et al. (8), where they were referred to as LUVs. There, an extrusion membrane with pores of about 100 nm (“nanometers”) in diameter created LUVs of about 123+/ ⁇ 35 nm. That distinguished them from small unilamellar vesicles “SUVs” that had a diameter of 34+/ ⁇ 30 nm.
  • phosphatidyl choline referred to in reference 8 was isolated from eggs
  • synthetic phosphatidyl cholines can also be used if they are in the liquid (or liquid crystal) state (i.e., not in the gel or solid state) at body temperature (likely if they have at least one double bond in the fatty acyl side-chains) yet are resistant to oxidation (do not have many double bonds).
  • An example is POPC.
  • LEVs constructed from POPC were used in the examples below.
  • the LEVs be composed of lipids that are liquid (or liquid-crystalline) at 37° C., often at 35° C., and even 32° C. Liposomes in the liquid-crystalline state typically accept and donate component molecules with LDL and related apoB-lipoproteins more efficiently than do liposomes in the gel (or solid) state. Because patients typically have a core temperature of about 37° C., liposomes composed of lipids that are liquid-crystalline at 37° C. are generally in a liquid crystalline state during treatment and, therefore, optimize remodeling of LDL and other harmful apoB-lipoproteins.
  • the vesicle is an LEV. It is preferred the mean diameter of the LEVs, be at least 50 nm, more preferably at least 100 nm. Preferably the mean diameter of the LEVs is not more than 1000 nm (1.0 mm), more preferably not more than 250 nm, most preferably not more than 150 nm. Unilamellar vesicles are preferred over multilamellar vesicles, to facilitate exposure of liposomal components to LDL and related apoB-lipoproteins, to maximize remodeling of these particles.
  • Highly preferred sizes are ones that do not alter liver metabolism to raise total plasma LDL concentrations (8).
  • the size of the liposomal vesicles may be determined by dynamic light scattering (DLS), quasi-elastic light scattering (QELS) (13), size-exclusion chromatography, electron microscopy, and other methods well-known in the art.
  • Average LEV diameter may, if desired, be reduced by sonication of formed LEVs and/or extrusion through membranes of smaller pore-sizes and/or high-shear technologies. Intermittent application of these methods may be alternated with DLS, QELS, or other assessments to optimize LEV formation.
  • the LEV compositions of the present invention also comprise a pharmaceutically acceptable carrier.
  • Many pharmaceutically acceptable carriers may be employed in the compositions of the present invention.
  • normal saline will be employed as the pharmaceutically acceptable carrier, typically buffered, such as a phosphate-buffered saline.
  • suitable carriers include, e.g., 0.4% saline, half-normal saline, 0.3% glycine, and the like, including glycoproteins for enhanced stability, such as albumin, apolipoproteins, globulin, etc.
  • These compositions may be sterilized by conventional, well-known sterilization techniques.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride.
  • the concentration of liposomes in the carrier may vary. Generally, the concentration will be about 20-500 mg of liposomal lipid per ml of carrier, usually about 50-200 mg/ml, and most usually about 100-200 mg/ml. Persons of skill may vary these concentrations to optimize treatment with different liposomal components or of particular patients. For example, the concentration may be increased to lower the fluid load associated with treatment. This may be particularly desirable in patients having atherosclerosis-associated congestive heart failure or severe hypertension. Alternatively, liposomes composed of irritating lipids may be diluted to low concentrations to lessen inflammation at the site of administration.
  • the liposomes will be administered via a peripheral vein for convenience.
  • the LEVs will be administered into a large central vein, such as the superior vena cava or inferior vena cava, to allow highly concentrated solutions to be administered into large volume and flow vessels.
  • the LEVs may also be administered via a variety of other routes that allow them access to plasma apoB-containing lipoproteins or to intra-arterial apoB-containing lipoproteins. In this sense, “access” can mean direct access or indirect access.
  • the mode of LEV administration is preferably selected from the group consisting of parenteral administration, intravenous administration, intra-arterial administration, intramuscular administration, subcutaneous administration, transdermal administration, intraperitoneal administration, intrathecal administration, via lymphatics, intravascular administration—including administration into capillaries, arteriovenous shunts, and vascular stents for long-duration release, rectal administration, administration via a chronically indwelling catheter, and administration via an acutely placed catheter.
  • the frequency of administration and the dose administered on each occasion will be chosen so as to use the minimum dose needed to achieve the maximum beneficial effect on a person without significant side effects. This choice will be facilitated by the use of an assessment system for measuring extent of aggregation of atherogenic lipoprotein particles and/or their susceptibility to aggregation and/or their retention in the arteries of a human or other animal.
  • assessments are discussed above in relation to the methods of the invention, specifically as to systems that can be used to determine whether the LEV dose should be modified.
  • LEVs can be co-administering LEVs with other medications.
  • use of LEVs to remodel LDL to be less susceptible to aggregation can be favorably combined with a statin, which will lower overall plasma concentrations of LDL.
  • Exemplary agents that can be combined with LEVs can be selected from the group consisting of an inhibitor of cholesterol synthesis, a statin, simvastatin, atorvastatin, rosuvastatin, a fibrate, an SGLT2 inhibitor, a GLP1 agonist, a DPP4 inhibitor, metformin, a weight-loss drug, a CETP inhibitor, a PCSK9 inhibitor, a cholesterol absorption inhibitor, ezetimibe, low-dose aspirin, an inhibitor of acetyl-CoA carboxylase (ACC), an inhibitor of ATP-citrate lyase (ACL), an LDL-lowering drug, a triglyceride-lowering drug, gemcabene, an inhibitor of sulfatase-2, an inhibitor of sulfatase-2 production or secretion, bempedoic acid, an inhibitor of the microsomal triglyceride transfer protein, an antisense oligonucleotide against APOB mRNA, an inhibitor
  • Said other medications can be administered either by their usual route (e.g., statins given by mouth) or they can be incorporated into the LEVs.
  • LDL Particles from LEV-Treated Hypercholesterolemic Mice are Far Less Susceptible to SMase-Mediated Aggregation than are LDL Particles from PBS-Treated Hypercholesterolemic Mice
  • This example is designed to show, in an in-vitro (test-tube) assay, that LDL from LEV-treated mice is far more resistant to SMase-mediated aggregation than is LDL from control (saline-treated) mice.
  • the assay of the susceptibility of LDL to aggregation was performed according to prior literature (9, 10, 16). The example is important because SMase-mediated LDL aggregation is expected to be a major contributor to atherosclerotic plaques associated with cardiovascular disease.
  • the procedure used to make the LEVs from POPC in this Example was the following: procedures were performed in a sterile biological cabinet, under purified atmosphere (e.g., HEPA-filtered air), with all surfaces and equipment cleaned and sterilized. Synthetic, pure, dry, granular POPC from Avanti Polar Lipids, Inc., was dispersed in sterile, hospital-grade phosphate-buffered saline (However, many different aqueous buffers can be used to manufacture liposomes) by vortexing, to make MLVs, at a concentration of 200 mg POPC per ml.
  • Synthetic, pure, dry, granular POPC from Avanti Polar Lipids, Inc. was dispersed in sterile, hospital-grade phosphate-buffered saline (However, many different aqueous buffers can be used to manufacture liposomes) by vortexing, to make MLVs, at a concentration of 200 mg POPC per ml.
  • the MLVs were extruded 10 times under medium pressures (250 to 300 psi) through two stacked polycarbonate filters (100-nm pore size) that had been fitted into a 10-mL water-jacketed thermobarrel Extruder (Lipex Biomembranes).
  • the LEVs were then filter-sterilized by passage through a 0.45- ⁇ m pore-size filter, and an aliquot was verified by endotoxin assay to be endotoxin-free, essentially endotoxin-free, or low-endotoxin (e.g., ⁇ 0.50 EU/ml).
  • mice Sixteen hypercholesterolemic human apoB 100 (huApoB 100 ) transgenic mice were randomly divided into two groups of eight mice each. Mice in one group were injected with LEVs at a dose of 1000 mg of POPC per kg of body weight, while mice in the other group were injected with an equivalent volume of PBS (phosphate-buffered saline) solution free of LEVs. The plasma was taken from each mouse one hour later. Each of these 16 plasma samples was raised to a density of 1.063 g/ml and then ultracentrifuged, a process that floats up VLDL, LDL, and, when present, LEVs.
  • PBS phosphate-buffered saline
  • the supernatant was subjected to size-exclusion chromatography through a Superose 6 column to separate VLDL and LEVs, which are large, from LDL, which is smaller. To ensure purity of the LDL, some of the LDL samples were passed a second time over the size-exclusion column. The 16 purified LDL samples were each brought to a standard concentration and then incubated with SMase for the indicated times (horizontal axis in FIGS. 1 and 2 ).
  • the LDL particles (100 ⁇ l, 1 mg/ml) were incubated in the wells of microtiter plates at pH 5.5 at 37° C. with human recombinant sphingomyelinase (hrSMase).
  • hrSMase human recombinant sphingomyelinase
  • the mean diameter of the aggregated LDL particles was determined by dynamic light scattering (DLS) at different time points during the incubation (10). The results obtained are summarized in FIGS. 1 and 2 .
  • Example 2 The 16 LDL samples from Example 1 were also subjected to compositional analyses. Lipids were extracted by the Folch procedure, under nitrogen, in the presence of lipid anti-oxidants, and then subjected to an automated, high-throughput tandem mass spectrometry procedure that was previously described in detail (15).
  • mice injected with LEVs mice injected with PBS.
  • the treatment of mice with a single injection of LEVs resulted in a decrease in the molar ratio of sphingomyelin to phosphatidylcholine (SM:PC) in the LDL of the mice.
  • SM:PC phosphatidylcholine

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